In the last several years, we have identified and characterized a new oncofetal antigen (OFA-I) on melanomas and other human tumors that induces humoral antibody response in cancer patients. Anti-OFA-I antibody found in human sera is of the IgM class. The disease-free interval of postoperative Stage II melanoma patients correlated with serum level of IgM anti-OFA-I. In this research, we propose to establish cultured human cell lines that secrete monoclonal antibody to OFA-I. Two methods will be used: Establishment of human B-lymphoblastoid cell lines that secrete high titer anti-OFA-I antibody, and establishment of human lymphocyte-myeloma hybrid cell lines. We have recently been successful in the production of human IgM antibody to OFA-I in vitro from cancer patients' peripheral blood lymphocytes after transformation by Epstein-Barr Virus (EBV). The peak titer was 1:64 at 6-7 weeks after the initiation of culture. In the proposed study, we will attempt to select permanent B-lymphoblastoid cell lines that produce a high titer of antibody to OFA-I. Rosetting techniques will be used to select lymphocytes specifically binding to OFA-I before and after transformation of cells by EBV. Culture conditions that promote preferential growth of IgM producers will also be investigated. Hybridoma techniques will be applied to the human myeloma cell line U266 which will be fused with human lymphocytes derived from patients with high titers of circulating anti-OFA-I antibody or from those immunized with an OFA-I positive tumor cell vaccine. The limiting dilution methods will be used to select clones secreting a maximum titer of monoclonal antibody to OFA-I. Antibody specificities will be determined using OFA-I positive melanoma target cell lines and HLA-matched, but OFA-I negative lymphoblastoid cell lines that are autologous to the melanoma lines. Two assays, the radioimmunoassay with I125 labeled protein A and the immune adherence absorption techniques, will be used to measure the antibody.